A novel system of genetic transformation allows multiple integrations of a desired gene in Saccharomyces cerevisiae chromosomes

Guerra, Odanir Garcia; Rubio, Ileana G.S.; Filho, Claudionor Gomes da Silva; Bertoni, R; Govea, Rute Cardoso dos Santos; Vicente, Elisabete José

doi:10.1016/j.mimet.2006.04.014

Cited by 14 publications

(21 citation statements)

References 33 publications

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“…However, even with the complete xylose consumption pathway integrated and overexpressed, the developed strain LVY27 consumes xylose poorly. Since an efficient expression of xylose isomerase is one of the main metabolic bottlenecks to use this pathway, the cassette containing the xylA was reconstructed using the δ LTR element, which could increase the number of integrations as described elsewhere36. Adaptive evolution of the engineered strain in xylose allowed a rapid improvement of the fermentative performance.…”

Section: Discussionmentioning

confidence: 99%

Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains

Santos

Carazzolle

Nagamatsu

et al. 2016

Sci Rep

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The development of biocatalysts capable of fermenting xylose, a five-carbon sugar abundant in lignocellulosic biomass, is a key step to achieve a viable production of second-generation ethanol. In this work, a robust industrial strain of Saccharomyces cerevisiae was modified by the addition of essential genes for pentose metabolism. Subsequently, taken through cycles of adaptive evolution with selection for optimal xylose utilization, strains could efficiently convert xylose to ethanol with a yield of about 0.46 g ethanol/g xylose. Though evolved independently, two strains carried shared mutations: amplification of the xylose isomerase gene and inactivation of ISU1, a gene encoding a scaffold protein involved in the assembly of iron-sulfur clusters. In addition, one of evolved strains carried a mutation in SSK2, a member of MAPKKK signaling pathway. In validation experiments, mutating ISU1 or SSK2 improved the ability to metabolize xylose of yeast cells without adaptive evolution, suggesting that these genes are key players in a regulatory network for xylose fermentation. Furthermore, addition of iron ion to the growth media improved xylose fermentation even by non-evolved cells. Our results provide promising new targets for metabolic engineering of C5-yeasts and point to iron as a potential new additive for improvement of second-generation ethanol production.

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Section: Discussionmentioning

confidence: 99%

Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains

Santos

Carazzolle

Nagamatsu

et al. 2016

Sci Rep

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“…However, the method was laborious because it involved several transformations and screening procedures to remove the selection marker. Co-transformation is also a technique that allows the construction of a food-grade yeast strain by using a vector carrying the gene of interest simultaneously with another plasmid carrying a selectable marker (Guerra et al, 2006). A drawback related to co-transformation is low transformation efficiency.…”

Section: Discussionmentioning

confidence: 99%

A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability

Guo

Zhang

et al. 2010

J. Zhejiang Univ. Sci. B

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Abstract:The aim of this work was to construct a novel food-grade industrial arming yeast displaying β-1,3-1,4-glucanase and to evaluate the thermal stability of the glucanase for practical application. For this purpose, a bi-directional vector containing galactokinase (GAL1) and phosphoglycerate kinase 1 (PGK1) promoters in different orientations was constructed. The β-1,3-1,4-glucanase gene from Bacillus subtilis was fused to α-agglutinin and expressed under the control of the GAL1 promoter. α-galactosidase induced by the constitutive PGK1 promoter was used as a food-grade selection marker. The feasibility of the α-galactosidase marker was confirmed by the growth of transformants harboring the constructed vector on a medium containing melibiose as a sole carbon source, and by the clear halo around the transformants in Congo-red plates owing to the expression of β-1,3-1,4-glucanase. The analysis of β-1,3-1,4-glucanase activity in cell pellets and in the supernatant of the recombinant yeast strain revealed that β-1,3-1,4-glucanase was successfully displayed on the cell surface of the yeast. The displayed β-1,3-1,4-glucanase activity in the recombinant yeast cells increased immediately after the addition of galactose and reached 45.1 U/ml after 32-h induction. The thermal stability of β-1,3-1,4-glucanase displayed in the recombinant yeast cells was enhanced compared with the free enzyme. These results suggest that the constructed food-grade yeast has the potential to improve the brewing properties of beer.

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“…Assim, os co-transformantes, células portadoras do gene de interesse (transgênicas) sem qualquer marcador de seleção positiva podem ser selecionados. As seqüências de DNA, a serem introduzidas na célula hospedeira, podem ser oligonucleotídeos (Yamamoto et al, 1992), plasmídeos lineares, circulares e fragmentos de DNA obtidos por PCR (Duno et al, 1999;Rubió 2001;Guerra et al, 2006); podendo ser empregadas moléculas diferentes em uma mesma cotransformação, que podem permanecer na célula, replicando-se de maneira autônoma, integrando-se ao genoma, ou recombinando-se in vivo entre elas ou com o plasmídeo de seleção (Prado e Aguilera, 1994). Dada a versatilidade da metodologia, a cotransformação tem sido amplamente utilizada na levedura S. cerevisiae para a substituição de um gene pelo seu alelo mutado (Camargo 1994e Camargo 2000 1.4 Transformação de S. cerevisiae Durante a transformação de células de levedura, pode acontecer do plasmídeo se integrar em regiões homólogas do DNA cromossomal.…”

Section: Transformação Genética De Saccharomyces Cerevisiaeunclassified

“…Estes elementos já foram usados como alvo de inserção de informações genéticas clonadas em linhagens S. cerevisiae haplóides de coleções de cultura (Garfinkel, 2005;Guerra et al, 2006); e de linhagens selvagens industriais (Guerra et al, 2006). …”

Section: Transformação De S Cerevisiae Por δ -Integraçãounclassified

“…O vetor que promove a δ -integração, pGEMTEasyδ, não contém qualquer marcador de seleção positiva, por isto é realizada uma cotransformação juntamente com o plasmídio pAJ50 (2µm, LEU2, G418 R ), que permite a seleção de transformantes de linhagens de coleção de culturas por complementação gênica e, de linhagens industriais, pelo marcador de resistência a geneticina (Guerra et al 2006). …”

Section: Transformação De S Cerevisiae Por δ -Integraçãounclassified

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Clonagem de um alelo do gene SPT15 em Saccharomyces cerevisiae para aumento da produção de etanol.

Dalmolin¹

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A novel system of genetic transformation allows multiple integrations of a desired gene in Saccharomyces cerevisiae chromosomes

Cited by 14 publications

References 33 publications

Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains

Unraveling the genetic basis of xylose consumption in engineered Saccharomyces cerevisiae strains

A food-grade industrial arming yeast expressing β-1,3-1,4-glucanase with enhanced thermal stability

Clonagem de um alelo do gene SPT15 em Saccharomyces cerevisiae para aumento da produção de etanol.

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